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Steelhead Oncorhynchus mykissthat fail to emigrate seaward after release from hatcheries, commonly referred to as “residuals,” can have negative impacts on natural populations ranging from competition and predation to interbreeding with returning anadromous adults. We investigated how age at release, size, and maturation status influenced the rate of residual production in hatchery summer‐run steelhead released from the Winthrop National Fish Hatchery (Methow River, Washington) between 2010 and 2015. Migration data from 21,598 individuals implanted with PITtags identified 1,783 residual steelhead expressing two distinct phenotypes: immature male and female parr; and precociously mature males. We found that age at release significantly affected the predominant residual phenotype. Age‐1 steelhead residuals (S1 rearing strategy) were dominated by smaller parr of both sexes (fish < 146 mm FL), while age‐2 residuals (S2 rearing strategy) were dominated by mature males, although both phenotypes were present in both S1 and S2 residual groups. Collections of residual steelhead in the Methow River indicated that parr phenotype residuals grew as well as natural‐origin juvenile O. mykiss, suggesting potential competition for food resources and habitat. Detections of PITtags within the Methow River basin indicated that precocious male residuals may overlap both spatially and temporally with previously documented spawning anadromous adults, posing a potential genetic management risk. Both residual phenotypes had poor overwinter survival, and only 1 of the 1,783 residual fish eventually returned to the Methow River as an anadromous adult. We conclude that the ecological and genetic consequences of residual steelhead far outweigh their potential contribution to anadromous production, and measures should be taken to reduce their production by changing hatchery rearing practices.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to near-physiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play a major role in stimulating primary ovarian follicle development and the transition into secondary growth.In vivo treatment of coho salmon with the androgen, 11-ketotestosterone, dramatically alters the ovarian transcriptome during primary oocyte growth, and promotes the completion of primary ovarian follicle growth.

AbstractSteelhead Oncorhynchus mykissexhibit significant life history plasticity, including variation in freshwater residence time, age of smoltification, and maturation that are influenced by exceeding specific thresholds in size or body energy stores. Fish that do not reach minimum thresholds for smoltification or exceed thresholds for maturation may “residualize” in freshwater. Understanding life history diversity is important in steelhead hatchery programs. The objective of this investigation was to characterize life history diversity in Hood River, Oregon, hatchery winter-run steelhead prior to release. We used both morphological and physiological indices to assess degree of smoltification in both sexes and maturation in males. Smoltification was categorized by measuring size, visual smolt index, and gill Na+/K+-ATPase activity. All females were determined to be immature. Male reproductive development required histological staging of testes. Other indices of maturation—including gonadosomatic index, plasma 11-ketotestosterone, and pituitary fshband lhband testis ifg3and amhgene expression—were also employed, with varying degrees of success. Five phenotypes were described: (1) immature male and female parr; (2) precociously mature male parr; (3) male smolts in early to mid-spermatogenesis that are likely to spawn after <1 year in the ocean; (4) males that might have attempted maturation, but failed; and (5) immature male and female smolts that could mature after >1 year in the ocean. Over three brood years, we estimated that 3–4% of the population could potentially residualize due to failure to smolt or precocious male maturation, 1–12% could mature after <1 year in the ocean (one salt male), and 84–95% could mature after >1 year in the ocean (two to three salt males and females). Based on previous studies conducted in the Hood River basin, the residualism rate estimated for this hatchery population is lower than that of naturally rearing Hood River steelhead.Received November 2, 2016; accepted February 14, 2017 Published online May 11, 2017

Steelhead Oncorhynchus mykissexhibit significant life history plasticity, including variation in freshwater residence time, age of smoltification, and maturation that are influenced by exceeding specific thresholds in size or body energy stores. Fish that do not reach minimum thresholds for smoltification or exceed thresholds for maturation may “residualize” in freshwater. Understanding life history diversity is important in steelhead hatchery programs. The objective of this investigation was to characterize life history diversity in Hood River, Oregon, hatchery winter-run steelhead prior to release. We used both morphological and physiological indices to assess degree of smoltification in both sexes and maturation in males. Smoltification was categorized by measuring size, visual smolt index, and gill Na+/K+-ATPase activity. All females were determined to be immature. Male reproductive development required histological staging of testes. Other indices of maturation—including gonadosomatic index, plasma 11-ketotestosterone, and pituitary fshband lhband testis ifg3and amhgene expression—were also employed, with varying degrees of success. Five phenotypes were described: (1) immature male and female parr; (2) precociously mature male parr; (3) male smolts in early to mid-spermatogenesis that are likely to spawn after <1 year in the ocean; (4) males that might have attempted maturation, but failed; and (5) immature male and female smolts that could mature after >1 year in the ocean. Over three brood years, we estimated that 3–4% of the population could potentially residualize due to failure to smolt or precocious male maturation, 1–12% could mature after <1 year in the ocean (one salt male), and 84–95% could mature after >1 year in the ocean (two to three salt males and females). Based on previous studies conducted in the Hood River basin, the residualism rate estimated for this hatchery population is lower than that of naturally rearing Hood River steelhead. Received November 2, 2016; accepted February 14, 2017 Published online May 11, 2017

Lean and siscowet morphotypes of lake trout Salvelinus namaycushin Lake Superior are thought to be genetically separate, but the reproductive isolating mechanism is unknown. The testicular and ovarian cycles and reproductive hormone levels of these morphotypes were determined from May to October in populations east and west of the Keweenaw Peninsula in southern Lake Superior. The gonadosomatic index (GSI) increased from August to October for lean and siscowet males and females east of the Keweenaw Peninsula and for siscowets west of the Keweenaw Peninsula. Circulating estradiol-17ß (E2) levels and ovarian GSIs increased simultaneously in females of both morphotypes. However, circulating 11-ketotestosterone (11-KT) levels in lean and siscowet males were not significantly elevated until October even though testicular GSIs increased by August. Transcripts of the follicle stimulating hormone (FSH) beta subunit (an indirect measure of FSH activity) increased in lean and siscowet males and females during August and September, when GSIs were increasing for both morphotypes. The seasonal changes in GSIs and hormone levels indicate that both lean and siscowet individuals in southern Lake Superior populations undergo reproductive maturation at the same time in the fall; therefore, reproductive timing does not appear to genetically isolate the morphotypes in these populations. A proportion of the sampled females (lean lake trout: 54%; siscowets: 42%) exhibited no increase in GSI from August to October, strongly suggesting that in any given year some proportion of the population does not reproduce. This was also observed in males but at a lower percentage (19–20%). Fish that did not have maturing gonads from August to October also had lower E2 and 11-KT levels than maturing fish. Fecundity measured for lean and siscowet lake trout was not statistically different and was similar to historical values.

AbstractLean and siscowet morphotypes of lake trout Salvelinus namaycushin Lake Superior are thought to be genetically separate, but the reproductive isolating mechanism is unknown. The testicular and ovarian cycles and reproductive hormone levels of these morphotypes were determined from May to October in populations east and west of the Keweenaw Peninsula in southern Lake Superior. The gonadosomatic index (GSI) increased from August to October for lean and siscowet males and females east of the Keweenaw Peninsula and for siscowets west of the Keweenaw Peninsula. Circulating estradiol-17β (E2) levels and ovarian GSIs increased simultaneously in females of both morphotypes. However, circulating 11-ketotestosterone (11-KT) levels in lean and siscowet males were not significantly elevated until October even though testicular GSIs increased by August. Transcripts of the follicle stimulating hormone (FSH) beta subunit (an indirect measure of FSH activity) increased in lean and siscowet males and females during August and September, when GSIs were increasing for both morphotypes. The seasonal changes in GSIs and hormone levels indicate that both lean and siscowet individuals in southern Lake Superior populations undergo reproductive maturation at the same time in the fall; therefore, reproductive timing does not appear to genetically isolate the morphotypes in these populations. A proportion of the sampled females (lean lake trout: 54%; siscowets: 42%) exhibited no increase in GSI from August to October, strongly suggesting that in any given year some proportion of the population does not reproduce. This was also observed in males but at a lower percentage (19–20%). Fish that did not have maturing gonads from August to October also had lower E2 and 11-KT levels than maturing fish. Fecundity measured for lean and siscowet lake trout was not statistically different and was similar to historical values.Received November 13, 2010; accepted May 26, 2011

Unilateral ovariectomy ULO, removal of one ovary is a powerful technique for studying aspects of reproductive physiology, including follicular recruitment and growth. To examine effects of ULO for the first time in a semelparous species, coho salmon Oncorhynchus kisutch were unilaterally ovariectomized during midvitellogenesis approximately 3 months before spawning. At termination of the study 79 days postsurgery, single ovaries of ULO fish were gravimetrically equivalent to paired ovaries of sham surgery, control fish. There was no evidence of recruitment of new vitellogenic follicles. Instead, the dramatic increase in ovary mass was attributable to hypertrophy of existing vitellogenic follicles 33 increase in volume and increased fecundity achieved through a greater than twofold reduction in follicular atresia. The composition of whole ovaries on a dry weight basis from ULO fish was greater in protein, but lower in lipid than that of control fish. Expressing the data on a per follicle basis, however, showed that follicles of ULO fish contained more protein, ash, water, and lipid. The results indicate that ULO of coho salmon induces compensatory hypertrophy of existing vitellogenic follicles, while maximizing fecundity through reduction of atresia. Thus, 3 months before spawning, coho salmon exhibit the ability to adjust final egg size and number when faced with significant depletion of ovarian follicles. This in vivosystem provides a platform for further study of physiological mechanisms regulating follicular growth and atresia, and the tradeoff between egg size and egg number. J. Exp. Zool. 309A:468–476, 2008. © 2008 WileyLiss, Inc.

Insulin-like growth factor-I (IGF-l) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-l on expression of genes encoding glycoprotein α (GPα), follicle-stimulating hormone (FSH) β, and luteinizing hormone (LH) β subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPα, FSHβ, and LHβ mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-l increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-l + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-l itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty. [ABSTRACT FROM AUTHOR]

AbstractThe Yakima River Spring Chinook Salmon Supplementation Project in Washington State is one of the most ambitious efforts to enhance a natural salmon population currently under way in the United States. Over the past 5 years we have conducted research to characterize the developmental physiology of natural and hatchery-reared wild progeny spring Chinook salmon Oncorhynchus tshawytschain the Yakima River basin. Fish were sampled at the main hatchery in Cle Elum, at remote acclimation sites, and, during smolt migration, at downstream dams. Throughout these studies, we characterized the maturational state of all fish using combinations of visual and histological analyses of testes, computation of gonadosomatic indices, and measurement of plasma 11-ketotestosterone (11-KT). We established that a plasma 11-KT threshold of 0.8 ng/mL can be used to designate male fish as either immature or precociously maturing approximately 8 months prior to final maturation (1–2 months prior to release as “smolts”). Our analyses revealed that 37–49% of the hatchery-reared males from this program undergo precocious maturation at 2 years of age and that a portion of these fish appear to residualize in the upper Yakima River basin throughout the summer. An unnaturally high incidence of precocious male maturation may result in the loss of returning anadromous adults, the skewing of female : male sex ratios, and ecological and genetic impacts on wild populations and other native species. As precocious male maturation is significantly influenced by the growth rate at specific times of year, in future studies we will alter maturation rates through seasonal growth rate manipulations.

The Yakima River Spring Chinook Salmon Supplementation Project in Washington State is one of the most ambitious efforts to enhance a natural salmon population currently under way in the United States. Over the past 5 years we have conducted research to characterize the developmental physiology of natural and hatchery-reared wild progeny spring Chinook salmon Oncorhynchus tshawytschain the Yakima River basin. Fish were sampled at the main hatchery in Cle Elum, at remote acclimation sites, and, during smolt migration, at downstream dams. Throughout these studies, we characterized the maturational state of all fish using combinations of visual and histological analyses of testes, computation of gonadosomatic indices, and measurement of plasma 11-ketotestosterone (11-KT). We established that a plasma 11-KT threshold of 0.8 ng/mL can be used to designate male fish as either immature or precociously maturing approximately 8 months prior to final maturation (1–2 months prior to release as “smolts”). Our analyses revealed that 37–49% of the hatchery-reared males from this program undergo precocious maturation at 2 years of age and that a portion of these fish appear to residualize in the upper Yakima River basin throughout the summer. An unnaturally high incidence of precocious male maturation may result in the loss of returning anadromous adults, the skewing of female : male sex ratios, and ecological and genetic impacts on wild populations and other native species. As precocious male maturation is significantly influenced by the growth rate at specific times of year, in future studies we will alter maturation rates through seasonal growth rate manipulations.

The effects of insulin-like growth factor I (IGF-I) and insulin on the function of coho salmon gonadotropes in vitro were investigated. Dispersed pituitary cells from immature coho salmon (Oncorhynchus kisutch) were incubated with IGF-I for 1, 3, 7, or 10 days, then incubated with salmon GnRH for an additional 24 h. Medium FSH content before and after GnRH treatment and intracellular FSH content after GnRH treatment were measured. Incubation of pituitary cells with IGF-I for 7 or 10 days increased GnRH-stimulated FSH release and remaining cell content, but did not affect basal release. To examine the specificity of the effects of IGF-I, we compared FSH release and cell content of FSH and LH after 10-day incubation with a range of concentrations of IGF-I or insulin. Incubation with physiological concentrations of IGF-I resulted in significantly higher GnRH-stimulated FSH release and remaining cell content of FSH and LH. Conversely, supraphysiological concentrations of insulin were required to produce more moderate effects on gonadotropin levels. These results suggest that elevation of gonadotropin levels by IGF-I may be one mechanism by which somatic growth and nutrition promote pubertal development in salmon.

Insulin-like growth factor-binding proteins (IGFBPs) may interfere with accurate measurement of plasma IGFs in radioimmunoassay (RIA). Although several simplified extraction methods for IGFs have been developed, these methods are not always validated for differing physiological states, developmental stages, and animal species. For teleost fish, neither the necessity of plasma extraction nor the validity of extraction methods for IGF RIA is widely established. We systematically examined the validity of acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation (AEC extraction), and SP-Sephadex extraction in RIA for salmon IGF-I using commercially available components (GroPep Pty Ltd). Displacement curves of plasma extracted by AE, AEC, and SP-Sephadex were parallel to those of the standard. Measured IGF-I levels in plasma from several developmental stages and under different physiological and experimental conditions were significantly increased by the extractions and comparable to those after acid-size exclusion chromatography (SEC). On Western ligand blotting using digoxigenin-labeled human IGF-I, the intensity of IGFBP bands remaining in plasma were reduced after extraction, although some IGFBPs remained. However, these residual IGFBPs did not interfere measurably with the RIA based on quantitative comparison of IGF-I levels with acid-SEC. We conclude that with this RIA extraction is necessary for measurement of salmon IGF-I in plasma since measured values were routinely lower in unextracted samples, and AE, AEC, and SP-Sephadex extractions are applicable to the IGF-I RIA using the commercially available components. Using the validated RIA for IGF-I, plasma IGF-I levels in nonmaturing and precociously maturing chinook salmon in spring were measured after AE extraction. During spring, nonmaturing and maturing fish fed and grew well, and plasma IGF-I level was significantly correlated with body weight in both fish. This result indicates that circulating IGF-I plays a key role in controlling growth in precociously maturing chinook salmon in spring as in nonmaturing fish.

Previous work has indicated that, during the process of gametogenesis in salmon, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are differentially synthesized and released. Although substantial information is available on the regulation of LH in many fish species, relatively little is known about the regulation of FSH biosynthesis and secretion or the regulation of two types of α subunit in salmon. In this study, the effects of salmon gonadotropin-releasing hormone (sGnRH) on in vitrosecretion of FSH, and α1, α2, LH β, and FSH β subunit gene expression were investigated in coho salmon (Oncorhynchus kisutch) using primary pituitary cell cultures. To quantify FSH β, LH β, α1, and α2 subunit transcript levels, a multiplex RNase protection assay (RPA) was developed. Probes for the β subunits of coho salmon FSH and LH were available from previous studies. To generate probes for the α subunit RPAs, α1 and α2 subunit cDNAs were cloned using reverse transcriptase PCR. Release of FSH and LH into cell culture medium was quantified by radioimmunoassays. The effects of sGnRH on gonadotropin release and gene expression were tested at two points during the spring (April and May) prior to spawning in the autumn; a period when plasma and pituitary FSH levels are increasing and females are in early stages of secondary oocyte growth. In both experiments, sGnRH increased steady-state mRNA levels of FSH β, α1, and α2, whereas LH β mRNA levels were not detectable. Secretion of FSH was stimulated by sGnRH in a concentration-dependent manner. Medium LH was not detectable in the first experiment (April) and was measurable only after sGnRH treatment in the second experiment (May). Control levels of medium FSH and transcripts for FSH β and α1 subunits increased approximately fourfold between April and May, whereas α2 transcript levels remained relatively constant, suggesting that the seasonal increase in FSH release may involve increased production of α1. Therefore, sGnRH has direct stimulatory effects on both secretion of FSH and FSH subunit biosynthesis, most likely due to increased transcription. However, alterations in rates of transcript degradation cannot be ruled out.

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17α-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17β production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20β-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.

The effects of long-term peripheral exposure to recombinant human leptin were tested in immature coho salmon under both fed and fasted conditions. We found that high circulating levels of human leptin did not alter growth, energy stores, gonad weight, pituitary content of follicle-stimulating hormone, or plasma levels of insulin-like growth factor-I, insulin, growth hormone, or thyroxine.

Prolactin (PRL) and two variants of growth hormone (GH), purified from pituitaries of striped bass (Moronesaxatilis) and its hybrid with white bass (M.saxatilis× M. chrysops) by gel filtration chromatography under alkaline conditions followed by reversed-phase high pressure liquid chromatography, appear similar between species. Both the minor (GH I) and the major (GH II) forms of purified GH appeared as single bands (Mr∼23,000) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, as did the purified PRL (Mr∼24,000). The molecular weights of GH II and PRL determined by MALDI TOF mass spectroscopy were 21.2 and 21.3 kDa, respectively. In Western blotting experiments, an antiserum against tilapia (Oreochromismossambicus) 24K PRL specifically recognized MoronePRL, while an antiserum against tilapia GH specifically recognized MoroneGH I and II. Chemical identities of the putative PRL and GH I were further confirmed by N-terminal peptide sequencing, while internal sequence analysis was performed on GH II because it was blocked at its N-terminus. Over a stretch of 29 amino acids, MoronePRL was found to be 76% identical to tilapia 24K PRL, 72% identical to tilapia 20K PRL, 72% identical to chum salmon (Oncorhynchusketa) PRL I, and 69% identical to eel (Anguillajaponica) PRL I. Alignment of the hybrid striped bass GH sequences with those of several other advanced marine teleosts indicated 75–85% sequence identity for GH I (40 amino acids) and 95–98% identity for GH II (45 amino acids). Biological activity of striped bass GH II was confirmed using a heterologous invitroassay of insulin-like growth factor I mRNA production by coho salmon (On. kisutch) hepatocytes. An invivobioassay, involving hypophysectomy of hybrid striped bass and treatment of the fish maintained in fresh water with homologous PRL, confirmed that the purified striped bass PRL was also bioactive.

Sexual maturation of Atlantic salmon (Salmo salar) male parr is a seasonally recurrent “all or none” response; either a fish matures fully or it does not mature. To study whether gonadal feedback on gonadotropic hormones, GTH I and GTH II, is involved in the control of maturation, previously mature Atlantic salmon male parr were either sham-operated or castrated in spring. They were then sampled during the onset of gonadal growth (late June–early July) or shortly before the breeding season (late September). In autumn, sham-operated males separated into two groups: nonrematuring males with low pituitary and plasma levels of both GTH I and GTH II, and those rematuring with high levels of gonadotropins. Castrated males had low GTH I and GTH II plasma and pituitary levels, similar to those of the nonrematuring fish, suggesting positive feedback mechanisms, separating the sham-operated fish into low and high GTH level groups. In the summer, plasma GTH II was nondetectable in all fish. Pituitary GTH II content was lower in nonrematuring, than in rematuring males and was even lower in castrated fish. In contrast, castration increased pituitary and plasma levels of GTH I in the summer, suggesting a negative feedback at this reproductive stage. There were no significant differences in immunoassayable levels of GTH I in plasma in rematuring and nonrematuring sham-operated males at this time. The control of rematuration is complex and may involve factors other than circulating GTH I levels, possibly with differences in gonadal sensitivity to GTH I.

Total and free insulin-like growth factor-I (IGF-I) levels were quantified in plasma from growth hormone (GH)-treated and fasted coho salmon. Total IGF-I was measured by radioimmunoassay after acid–ethanol extraction and free IGF-I was separated from protein-bound IGF-I using ultrafiltration by centrifugation. Total and free IGF-I increased in plasma after GH treatment and decreased after fasting. The level of free IGF-I, however, was maintained at approximately 0.3% in both experiments. Unsaturated binding activity in plasma for IGF-I was assessed by incubation with 125I-recombinant salmon IGF-I (125I-sIGF-I). Although there was no difference in binding activity between GH-treated and control fish, fasted fish showed higher binding activity than did fed fish, suggesting induction of unsaturated binding protein by fasting. IGF-binding protein (IGFBP) bands were observed in plasma of coho salmon by Western ligand blotting using 125I-sIGF-I. A low-molecular-weight (22 kDa) band was clear in fasted fish but not detectable in fed fish. The IGFBP band, which has molecular weight similar to that of human IGFBP-3 (41 kDa), was more intense in GH-treated fish than in controls. The molecular distribution of IGF-I in plasma was examined by gel filtration under neutral conditions. Most IGF-I was eluted around 40 kDa. This result suggests that the major form of bound IGF-I in the circulation of coho salmon may be in a 40-kDa binary complex rather than in a 150-kDa ternary complex, as in mammals.

Both positive and negative feedback on the (hypothalmus)– pituitary–gonad axis occur in salmonids. The aim of the present study was to investigate the role of different androgens, and in particular the involvement of aromatization of androgens to estrogens in feedback mechanisms. Previously mature Atlantic salmon,Salmo salar,male parr were studied in two experiments. In the first experiment, intact fish were implanted with Silastic capsules filled with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in spring and sampled in the autumn when the rematuring males were starting to display running milt. In the second experiment, castrated males were implanted with capsules containing ATD, the androgens testosterone (T) and 11-ketoandrostenedione (11KA), or ATD and T combined in spring. These fish were sampled in the summer when rematuring fish were starting to show signs of gonadal growth. Pituitary and plasma gonadotropins (GTH I and GTH II) were studied using radioimmunoassay. In autumn, ATD treatment reduced pituitary and plasma GTH II levels. In summer, GTH II was consistently nondetectable in plasma. Castration diminished pituitary GTH II content. Treatment with T increased pituitary GTH II content, an effect that was attenuated when T treatment was combined with ATD. All these results are consistent with the presence of an aromatase-dependent positive feedback of T on GTH II. 11KA also had a stimulatory effect on GTH II, although weaker than that of T. Testicular size and spermiation was reduced by ATD in autumn; the latter of these results is likely to be due to the inhibitory effect of ATD on GTH II. Positive effects of ATD on plasma GTH I were found in autumn, indicative of an aromatase-dependent negative feedback in this phase. On the other hand, castration increased plasma and pituitary GTH I levels in summer, indicating that the gonads in this phase exert a predominantely negative control of GTH I. In summer, negative effects of T on GTH I pituitary levels were not suppressed, but were rather enhanced, by the combined treatment with ATD. Furthermore, plasma GTH I levels were lower after treatment with T in combination with ATD than with T or ATD separately. These negative effects of T were not diminished by ATD, so that they are nonaromatase-dependent. Furthermore, since they were actually more pronounced in the presence of ATD it is suggested that there is also a positive aromatase-dependent feedback component in this phase. In addition, 11KA had a negative effect on plasma and pituitary GTH I in castrated previously mature males. Thus, GTH I secretion is controlled by both aromatase-dependent and nonaromatase-dependent feedback effects, of which at least the former may be positive or negative depending on season. In summary, the feedback control of GTH I appears to be more complex than that of GTH II.

Atlantic salmon, Salmo salar, mature male parr were implanted with testosterone (T) in small (T3) or large (T10) Silastic capsules in the breeding season or at its end in November or December, in order to find out whether the postbreeding decline in 17α,20β-dihydroxy-4-pregnene-3-one (17,20β-P) and milt production is a consequence of declining T levels. In the first of three experiments, fish were sampled the following January and March, whereas in the second and in the third they were sampled in April. Pituitary and plasma concentrations of gonadotropic hormone (GTHs) I and II and plasma levels of T, 11-ketotestosterone (11-KT) and 17,20β-P were measured by radioimmunoassays, and the testes were examined histologically. Administration of T prolonged the period in which running milt was present, suppressed Sertoli cells, and prevented the postbreeding decline in testes weight in experiment two. The postbreeding decline in plasma 17,20β-P levels was diminished by T10 in experiment one, and by both T3 and T10 in experiment two. The similar decline in 11-KT levels was not influenced by T treatment (only studied in experiment one). T treatment also prevented a decline in pituitary GTH II content (in experiments two and three) and in plasma GTH II levels (only studied in experiment three). However, pituitary GTH I content was not influenced (experiment two and three), whereas plasma GTH I levels (only studied in experiment three) were suppressed by T. To summarize, T treatment prevents postbreeding decline in 17,20β-P levels, probably via a stimulation of GTH II secretion. J. Exp. Zool. 284:425–436, 1999. © 1999 Wiley-Liss, Inc.

Receptors for two salmon gonadotropins, GTH I and GTH II, were localized by use of in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation. The results in both sexes revealed the presence of two types of GTH receptors: type I (GTH-RI), which interacts with both GTHs, and type II (GTH-RII), which interacts specifically with GTH II. GTH-RI was found at all stages of spermatogenesis examined and was localized on cells that were most likely Sertoli cells; however, it could not be determined whether GTH-RI was also localized on Leydig cells. In contrast, GTH-RII was found only in Leydig cells in the testis from a spermiating fish. In the vitellogenic ovary, GTH-RI was localized in the thecal layer and intensely on granulosa cells; in the preovulatory follicle, in contrast, GTH-RI was found in the thecal layer and in interstitial connective tissue, but not in the granulosa layer. Among all the stages of oogenesis examined, only granulosa cells of the preovulatory follicle exhibited GTH-RII. The appearance of GTH-RII coincides well with the increase in plasma levels of GTH II that occurs during final oocyte maturation and spermiation in coho salmon. The nature, distribution, and timing of appearance of these two receptors can explain, at least in part, the results of previous studies on steroidogenic activities of the two GTHs. The present study also suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development.

The first appearance of adenohypophysial cells in the developing rainbow trout embryo was studied by immunocytochemistry. Antibodies generated against the β subunits of coho salmon gonadotropins (GTH I and GTH II), the β subunit of human thyroid stimulating hormone (TSH), chum salmon prolactin (PRL), chum salmon growth hormone (GH), and synthetic α-MSH were used as immunocytochemical probes. The pituitary anlage was first recognized at developmental stage 21 (18 days postfertilization). At this stage, immunoreactive (ir) PRL and α-MSH cells were identified in the rostral and caudal regions of the pituitary, respectively. Cells containing ir-GH and ir-adrenocorticotropin (ACTH) appeared at stage 28 (35 days postfertilization). At the last stage before hatching (stage 29, 42 days postfertilization) ir-TSH cells were identified, Ir-GTH I cells were first observed in the pituitary at stage 32 (15 days after hatching), when mitosis of gonadal germ cells also was observed. At stage 35 (35 days after hatching), meiotic figures were first observed in a few germ cells of some fish indicating that gonadal sex differentiation probably begins at about this time. Cells containing ir-GTH II were not apparent in the pituitary at any stage of embryonic or larval development (up to stage 35) and were not present in pituitaries of trout at 6 months of age when the ovaries were in the perinucleolus stage and when only spermatogonia, but no spermatocytes were present in the testis. These observations suggest that GTH I, but not GTH II, may regulate initial gonadal growth and development in the embryonic and larval rainbow trout. The appearance of other pituitary hormones early in development invites speculation regarding their possible functions at this time. Copyright 1993, 1999 Academic Press

The circulatory half-life of the mammalian glycoprotein hormone lutropin is controlled by its unique Asn-linked oligosaccharides, which terminate with the sequence SO4-4-GalNAcβ1,4GlcNAc. A cluster of basic amino acids essential for recognition of the α subunit by the glycoprotein hormone:N-acetylgalactosaminyltransferase is located within two turns of an α helix (Mengeling, B. J., Manzella, S. M., and Baenziger, J. U.(1995) Proc. Natl. Acad. Sci. U. S. A.92, 502-506). The amino acids within this region are virtually invariant in the α subunits of all vertebrates, indicating that the recognition determinant utilized by the N-acetylgalactosaminyltransferase has been conserved in species ranging from teleost fish to mammals. We demonstrate that the glycoprotein hormone:N-acetylgalactosaminyltransferase and the N-acetylgalactosamine-4-sulfotransferase responsible for the synthesis of these unique sulfated oligosaccharides are expressed in the pituitaries of vertebrates ranging from teleost fish to mammals. Furthermore, we show that Asn-linked oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAc are present on the α and β subunits of the salmon glycoprotein hormone GTH II. Asn-linked oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAc are unique structural features of the glycoprotein hormones that have been conserved during vertebrate evolution, suggesting they are critical for the expression of hormone biologic activity.

Previous studies from our laboratory have shown that salmon gonadotropins GTH I and GTH II have similar effects on testicular steroidogenesis in vitro. To determine whether the relative potencies of GTH I and GTH II changed during late stages of spermatogenesis, the effects of GTH I and GTH II on in vitro steroid production by testicular tissue from fish in stages IV to V of spermatogenesis were examined. Fragments of testicular tissue were incubated with GTH I and GTH II for 18 h at 15°C. The in vitro production of 11-ketotestosterone (11-KT), testosterone (T), 17α-hydroxyprogesterone (17OH-P), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was determined by RIA. GTH I and GTH II were equipotent in stimulating the production of 11-KT, T, and 17,20β-P (17OH-P was not detectable) by testicular tissue in stage IV of spermatogenesis. The sensitivity of testicular tissue to the steroidogenic effects of GTH II increased as spermatogenesis progressed. In contrast, the sensitivity of testicular tissue to the effects of GTH I on 17,20β-P production declined from stages IV to V, while the sensitivity of testicular tissue to the effects of GTH I on 11-KT production remained unchanged. These changes in sensitivity to GTH I and II are consistent with previously reported changes in GTH receptors in salmon testis and support the hypothesis that GTH I and GTH II may have different roles during spermatogenesis.

The possible existence of distinct receptors for salmon gonadotropins (GTH I and GTH II) and the distribution of the receptor(s) were studied through examination of the binding of coho salmon (Oncorhynchus kistuch) GTH I and GTH II to membranes from thecal layers and granulosa cells of salmon ovaries. Purified coho salmon gonadotropins were iodinated by the lactoperoxidase method. Crude membrane preparations were obtained from thecal layers, granulosa cells, and whole ovaries of coho salmon in the postvitellogenic/preovulatory phase. Binding of 125I-GTH I to membranes from thecal layers, granulosa cells, and whole ovaries, and binding of 125I-GTH II to thecal layer cell membranes could be inhibited by both GTHs, but GTH I was more potent than GTH II. In contrast, GTH II was more potent than GTH I in inhibiting 125I-GTH II binding to membranes from granulosa cells and whole ovaries, but the inhibition curves were not parallel. Scatchard plot analysis suggested that there was a single type of receptor in the thecal layers for both GTHs, whereas in the granulosa cells there was more than one type of receptor for both GTHs. Based on these results, a two-receptor model for the postvitellogenic/preovulatory salmon ovary is proposed with the following features: 1) there are two types of gonadotropin receptors in the salmon ovary, type I and type II; 2) the type I receptor binds both GTHs, but with higher affinity for GTH I, whereas the type II receptor is highly specific for GTH II and may have only limited interaction with GTH I; and 3) the type I receptor is present in both thecal cells and granulosa cells, whereas the type II receptor is present in granulosa cells.

At 6 months of age the first spermatozoa appear in the testes of the African catfish considered to be adolescent, since the development to adulthood (12 months of age) is accompanied by further morphological and functional differentiation of Leydig cells. There are increasing plasma levels of 11-ketotestosterone (11-KT) and an increasing responsiveness to luteinizing hormone (LH) of testicular androgen secretionin vitro.Whether treatment of adolescent males with key hormones of the brain–pituitary–gonad axis [gonadotropin-releasing hormone (GnRH), LH, and 11-KT] affects the testicular steroidogenic response to a challenge with LHin vitro7 days later has been investigated. Injection of GnRH (2.5 μg chicken GnRH-II per kilogram of body weight), LH (25 μg/kg), or a high dose of 11-KT (50 μg/kg) down-regulated basal and LH-stimulated testicular androgen secretion to a minimum of 35% of control values. Treatment with LH was, moreover, associated with changes in the ultrastructure of Leydig cell mitochondria which were either swollen and had a less electron-dense matrix or showed an elongated shape. Conversely, a moderate dose of 11-KT (20 μg/kg) enhanced LH-stimulated, but not basal, androgen secretionin vitroto a maximum of 190% of control values. In view of the generally low LH plasma levels and of the steadily increasing 11-KT plasma levels during puberty, 11-KT may be involved in the up-regulation of the testicular steroidogenic capacity observed during development to full maturity.

A specific homologous radioimmunoassay (RIA) for measurement of insulin-like growth factor-I (IGF-I) in plasma of salmonid and a few non-salmonid fish species was developed using recombinant coho salmon IGF-I (rsIGF-I) as tracer and standard, and antiserum against this peptide raised in rabbits. The minimum detection level of IGF-I was 1.5 ng/ml and linearity was obtained in a range from 1.5 to 23 ng/ml. No cross-reaction was detected in the salmon IGF-I RIA with mammalian growth factors, salmon pituitary hormones, salmon or mammalian insulin, or any peptide in rat plasma. Although salmon IGF-I has high sequence similarity to mammalian IGF-I, it did not cross-react with anti-human IGF-I serum in human RIA and serial dilutions of plasma from salmon were not parallel to the human IGF-I standards in this assay system. In contrast, dilution curves for plasma of salmonids, such as coho (Oncorhynchus kisutch), Atlantic (Salmo salar), and sockeye (O. nerka) salmon, rainbow trout (O. mykiss), some other teleost fish, such as tilapia (Oreochromis mossabmica), carp (Cyprus carpio), eel (Anguilla rostrata), Atlantic halibut (Hippoglossus hippoglossus), and agnathan, the sea lamprey (Petromyzon marinus), assessed in salmon IGF-I RIA were parallel to the rsIGF-I standards. Acid-ethanol extraction of plasma samples altered the molecular weight, but not the quantity, of immunoreactive IGF-I, implying that IGF-I binding proteins in salmon plasma do not affect the performance of the salmon IGF-I RIA. Gel filtration of nonacidified plasma on a Sephadex G-75 superfine column produced two immunoreactive IGF-I peaks of molecular weights of approximately >70 k and 7 kDa, whereas acidification of plasma increased the relative amount of the 7-kDa peak (IGF-I) and the >70-kDa peak disappeared. The recoveries of rsIGF-I added to extracted or nonextracted plasma were 97.4 and 94.9%, respectively. Inter- and intraassay coefficients of variation were 3.6 and 3.3%, respectively. Plasma IGF-I levels in coho salmon smolts were 117.4 ± 19.1 ng/ml as compared to IGF-I levels in parr (45.3 ± 2.5 ng/ml) or in adult fish (45.2 ± 5.4 ng/ml) measured in the same assay. Injection of salmon growth hormone, but not prolactin or somatolactin, caused a significant and dose-dependent elevation of plasma IGF-I levels, while either fasting or injection of streptozotocin led to a significant decline in systemic IGF-I. We conclude that the salmon IGF-I RIA reported in this study is valid for measurement of IGF-I levels in salmonids and, likely, in a variety of other fish species.

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologousin vivobioassay and by immunoblots using anti-human TSH β-subunit antisera.In vivotreatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH β subunit has 56% sequence identity to that of human TSH β subunit and is identical to the deduced amino acid sequence of trout TSH β subunit. The N-terminal sequence (25 residues) of the salmon TSH α subunit is identical to gonadotropin α-II subunit. Molecular sizes of the α and β subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS–PAGE. Antiserum generated against salmon TSH, which was preadsorbed with α subunit using an α-subunit affinity column, detected only salmon TSH β subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH β subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I β and GTH II β subunits, α subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally,in vivoinjection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.

Studies of both salmon and trout have indicated that the levels of gonadotropins, GTH I and GTH II, in the pituitary and peripheral circulation vary during the reproductive cycle. To evaluate the possible feedback relationship between the gonads and pituitary GTH secretion, we studied the effects of bilateral gonadectomy on plasma levels of GTH I and GTH II in coho salmon,Oncorhynchus kisutch.During late spermatogenesis in males and late vitellogenesis in females, plasma GTH I levels increased significantly after gonadectomy, approximately 6- and 5-fold over presurgery levels at 3 and 14 days after surgery, respectively, and then declined to near presurgery levels by Day 17. No change in GTH I levels occurred in sham-operated fish. In all groups, GTH II levels were nondetectable and did not change significantly up to 17 days postsurgery. In males gonadectomized during spermiation, plasma GTH I levels increased significantly, approximately 10-fold over presurgery levels by 7 days postsurgery, and remained elevated thereafter. In contrast to the males in late spermatogenesis, the spermiating fish had detectable levels of GTH II (2–3 ng/ml), and significant elevations in plasma GTH II levels (approximately 60-fold) were observed 7 days after gonadectomy. These experiments demonstrate that the gonads exert negative feedback effects on secretion of both GTH I and GTH II, but the effect varies seasonally and the nature of the specific factor(s) from the gonads that inhibits and/or stimulates GTH production and secretion remains to be clarified.

Mature Atlantic salmon (Salmo salar) male parr were implanted in June and August with Silastic capsules filled with the progestin 17α,20β-dihydroxy-4-pregnene-3-one (17,20β-P) or with empty capsules. Experiments were terminated in September, when natural androgen levels are at their highest. Radioimmunoassay (RIA) showed that treatment raised plasma levels of 17,20β-P, whereas the content of gonadotropic hormones (GTH I and II) in the pituitary and plasma levels of the androgens testosterone (T) and 11-ketotestosterone (11-KT) were not influenced. Testicular fragments from mature salmon parr were incubated with different levels of 17,20β-P in combination with T or 17α-hydroxy-4-pregnene-3-one as precursors. A very high level of 17,20β-P (3,000 ng/ml) suppressed 11-KT production, whereas 3, 30, or 300 ng/ml were without effect. Thus, in these experiments physiological levels of 17,20β-P did not suppress androgen 11-KT production or alter pituitary gonadotropin levels, suggesting that some other mechanism is responsible for the natural decline in androgen levels when 17,20β-P levels rise at spawning. J. Exp. Zool. 277:66–71, 1997. © 1997 Wiley-Liss, Inc.

Investigations of hypothalamic regulation of fish thyrotropin (TSH) secretion and subsequent thyroid activity have been impeded by the lack of a reliable assay for TSH. Using a recently developed radioimmunoassay (RIA) for coho salmon TSH we employed anin vitropituitary cell culture technique to examine regulation of TSH secretion by corticotropin-releasing hormone (CRH) family peptides [ovine CRH (oCRH), carp urotensin I (UI), and frog sauvagine (SV)] as well as thyrotropin-releasing hormone (TRH), salmon growth hormone-releasing hormone (sGHRH), and salmon gonadotropin-releasing hormone (sGnRH). At concentrations of 0.01 to 100 nM, TRH, sGHRH, and sGnRH did not stimulate TSH secretion from coho salmon pituitary cells. However, at these same concentrations, both oCRH and SV caused a significant and concentration-dependent increase in TSH secretion; whereas, UI was highly stimulatory at all concentrations tested. In a related experiment we examined the effect of α-helical CRF(9–41)on oCRH-stimulated TSH release by pituitary cells. α-Helical CRF(9–41)is an analogue of CRH that has been shown by others to antagonize the adrenocorticotropic hormone (ACTH)-releasing activity of CRH in goldfish. Preincubation of cells with 1 μM α-helical CRF(9–41)for 4 h caused a significant suppression of the TSH-releasing activity of oCRH at 1.0 and 10 nM concentrations. The results of these experiments demonstrate the potency of a CRH-like peptide in the hypothalamic regulation of TSH in fish and reveal similarities in the inhibition of the response of both the thyroid and interrenal axis of fish to α-helical CRF(9–41).

In teleosts, ovarian steroidogenesis is under the control of two gonadotropic hormones, GTH I and GTH II, that are structurally and functionally homologous to FSH and LH. The intracellular mechanisms by which GTH I and GTH II stimulate steroidogenesis in the teleost ovary are not well understood. The purpose of this study was to investigate the involvement of the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/Ca 2+signaling pathways in the steroidogenic actions of GTH I and GTH II in the ovary of the brook trout (Salvelinus fontinalis). The cAMP/PKA pathway mediated the actions of GTH I before germinal vesicle breakdown (preGVBD) and GTH II after GVBD and before ovulation (preOV). Experimental increases in intracellular cAMP concentration mimicked the steroidogenic effects of GTH I and GTH II, and an antagonistic analog of cAMP partially blocked them. In addition, GTH I and GTH II stimulated the production of cAMP in preGVBD and preOV follicles, respectively. Activation of the PKC/Ca2+pathway by a phorbol ester or a Ca2+ionophore blocked the GTH I- and GTH I-induced steroid production, whereas inhibition of PKC by specific inhibitors potentiated the effects of GTH I. These results suggest that the cAMP/PKA signaling pathway mediates the stimulatory effects of GTH I and GTH II on steroidogenesis, and they also suggest the additional involvement of the PKC/Ca2+signaling pathway in modulating the actions of gonadotropins in brook trout ovarian follicles.

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of α and β subunits with N-terminal amino acid residues of Tyr, Gly (α, β of GTH I) and Tyr,Ser (α, β of GTH II). Coho salmon GTH I-β and GTH II-β differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-β and GTH II-β, respectively. Specific rabbit antisera to the β subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.

Somatolactin (SL), a recently identified teleost pituitary hormone which is a member of the growth hormone/prolactin family, was isolated from pituitary tissue of Atlantic halibut (Hippoglossus hippoglossus). Pituitary proteins were extracted in ammonium bicarbonate (pH 7.8), fractionated using gel filtration chromatography, and purified using reversed-phase high-performance liquid chromatography. Halibut SL was identified on the basis of molecular size (determined by gel electrophoresis and mass spectroscopy), cross-reactivity of the putative hormone with antisera to cod SL, and N-terminal amino acid sequence. Polyclonal antibodies to purified halibut SL were raised in rabbits, and a radioimmunoassay (RIA) was developed for measurement of plasma concentrations of SL using purified halibut SL as a standard. The RIA was tested in several flatfish species including Pacific halibut (Hippoglossus stenolepis), English sole (Pleuronectes vetulus), and rock sole (Lepidopsetta bilineata). The assay was specific for SL as indicated by absence of cross-reactivity with Atlantic halibut growth hormone, prolactin, and GTH α subunit. Dilutions of plasma and pituitary extracts from Pacific halibut, English sole, and rock sole were parallel to the Atlantic halibut SL standard curve, indicating that the assay is valid for a range of flatfish species. Using halibut SL antiserum, SL was localized in the pars intermedia of English sole pituitary, where it has been identified in previously examined teleost species. The RIA was used to measure plasma levels of SL in Atlantic halibut and English sole during reproductive development, and in English sole subjected to various types of environmental stressors, including handling and crowding. In both sole and halibut, plasma SL concentrations remained relatively constant throughout gonadal development, but dropped during or following ovulation. Plasma SL levels in English sole tended to increase in response to acute stress, in parallel with plasma cortisol levels.

Summary.Hysterectomy on or after Day 10 resulted in a quick return of pseudopregnant rats to oestrus but the interval to oestrus in pregnant rats was significantly longer (P< 0·01), indicating that the gravid uterine horn interfered with uterine-induced luteolysis by Day 10.When pseudopregnant rats were injected with extracts of conceptuses or pregnant rat serum (PRS), samples collected on Days 10 or 12 of pregnancy delayed the return to oestrus (P< 0·01 for conceptus extracts, P< 0·05 for PRS) and serum progesterone concentrations on Day 13 of pseudopregnancy were higher (P< 0·01) than in controls. Bioassays utilizing mammary development or inhibition of oestrus in virgin cyclic rats as endpoints failed to detect lactogenic activity in Day 10 conceptus extracts or PRS, and radioreceptor assays using bovine prolactin (NIH-B5) as a standard showed that Day 10 extracts had only about 2–3% of the lactogenic activity found in Day 12 extracts. Injections of dilutions of Day 12 extracts with 2 or 4 times the lactogenic activity as Day 10 extracts did not extend pseudopregnancy. These results suggest that by Day 10 the conceptus produces a factor capable of interfering with luteolysis and that this factor is not placental lactogen.

In this study, receptors for insulin and insulin-like growth factor I (IGF-I) in isolated theca-interstitial layers and granulosa cells of the coho salmon preovulatory ovary were characterized, and the effects of IGF-I on ovarian steroidogenesis were examined. Specific receptors for insulin and IGF-I were found in granulosa and theca-interstitial layers. In both follicular layers, IGF-I receptors were greater in number and higher in affinity than insulin receptors. The effects of IGF-I onin vitroproduction of testosterone (T) and 17α-hydroxyprogesterone (17OH-P) by theca-interstitial layers and of 17β-estradiol (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) by granulosa cell layers were evaluated during the preovulatory period. Both human and salmon recombinant IGF-I inhibited the basal and GTH II-stimulated T and 17OH-P production by theca-interstitial layers throughout the preovulatory period. In contrast, IGF-I stimulated the production of both E2and 17,20β-P by granulosa cell layers prior to germinal vesicle breakdown (GVBD) but only stimulated the production of 17,20β-P by granulosa cell layers after GVBD. The inhibitory effects of IGF-I on steroid production by the theca-interstitial layer and the opposite stimulatory effects on steroid production by the granulosa cell layer, coupled by the presence of specific IGF-I receptors in both follicular layers, suggest that IGF-I may play a role in the regulation of steroidogenesis in the preovulatory coho salmon ovary.

The ability of coho salmon gonadotropins GTH I and GTH II to stimulate testicular production of 11-ketotestosterone (11-KT) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) in vitro, as well as the involvement of cyclic AMP (cAMP) as a mediator of the actions of GTH I and GTH II, were investigated in maturing male coho salmon. Testicular tissue was incubated in the presence or absence of the test substances at 15°. Both GTH I and GTH II stimulated the in vitro production of 11-KT and 17,20β-P in a concentration- and time-dependent manner. In testicular tissue at stage IV of spermatogenesis, GTH I and GTH II stimulated the in vitro production of steroids with similar potency. The steroidogenic activity of GTH I and GTH II appeared to be mediated, at least in part, by cAMP as indicated by the ability of dibutyryl cAMP and forskolin to mimic the steroidogenic actions of GTHs, and the similar ability of GTH I and GTH II to increase intracellular cAMP levels in the testicular tissue. The calcium ionophore A23187 stimulated basal but not GTH-stimulated production of steroids in vitro. In addilion, differential effects of a protein kinase C inhibitor, H-7, on 11-KT and 17,20β-P production were found. Basal and GTH-stimulated production of 11-KT were inhibited by H-7; whereas basal and GTH-stimulated production of 17,20β-P were stimulated by H-7. These results suggest that protein kinase C may also be involved in the steroidogenic actions of GTH I and GTH II. Copyright 1993, 1999 Academic Press

Abstract: The salmonid pituitary produces two chemically distinct gonadotropins (GTHI and GTHII). Ultrastructural characteristics of GTHI- and GTHII-producing cells were studied in the trout pituitary with electronmicroscopic immunocytochemistry using antisera against salmon GTHIβ- and IIβ-subunits. In pituitaries from vitellogenic fish, GTHI-cells were characterized by numerous dilated cisternae of the granular endoplasmic reticulum (GER) and a small number of Iβ-positive granules (diameter, 100–300 nm), whereas GTHIIβ-immunoreactivity was found on granules (diameter, 200–400 nm) and large globules (diameter, 500–4000 nm) in apparently different cells (GTHII-cells). Distinct cellular distributions of GTHI and GTHII were maintained during gametogenesis, although morphological characteristics of GTHI- and GTHII-cells overlapped each other due to changes in number and size of the granules, globules and cisternae of the GER. Interestingly, the globules in the GTHI-cells were immunonegative for GTHIβ, although in the GTHII-cells they were always stained with GTHIIβ-antiserum. These results confirm that GTHIβ and GTHIIβ are synthesized in distinctly different cell-types in the salmonid pituitary and indicate that morphological characteristics cannot be used to distinguish these two cell-types.

Abstract: Somatolactin (SL) is a novel pituitary hormone recently characterized in several fish species. Structural analyses have shown that SL belongs to the growth hormone/prolactin family, and that it is a highly conserved protein. SL is synthesized by the periodic acid/Schiff-positive cells in the pars intermedia, but has an as yet unidentified function(s). We have recently developed a homologous radioimmunoassay for coho salmon SL and measured plasma levels of SL during two stages of the coho salmon life cycle, smoltification and sexual maturation. During smoltification, plasma levels of SL changed almost in parallel with plasma levels of thyroxine; levels increased as morphological indices of smoltification appeared and decreased as smoltification was completed. Following this period, SL levels remained low until the spring prior to spawning. In a separate study, plasma levels of SL were measured in sexually maturing coho salmon that remained in fresh water throughout their life cycle. During the year of sexual maturation, plasma levels of SL gradually increased from the spring onward, reaching peak levels at the time of spawning in November and December. These data are similar to those previously reported for sexually maturing coho salmon that were maintained in seawater prior to spawning (Rand-Weaver et al. 1992). Therefore, increases in plasma SL levels occurred in sexually maturing fish irrespective of whether they were maintained in fresh water or seawater. Peak levels at spawning were higher than those observed during smoltification. Possible roles for SL in metabolism and reproduction are discussed.

Abstract: The present study was designed to obtain basic endocrine information on GTH I and GTH II in previtellogenic and prespermatogenic coho salmon (immature). Levels of GTH II in pituitary extracts were 6.5 2.0 and 6.7 2.0 pg/μg pituitary protein in male and female fish, respectively. In contrast, the pituitary content of GTH I was approximately 100-fold higher than GTH II (1.302 .22 and 1.173 .21 ng/μg pituitary protein in male and female fish, respectively). Plasma levels of GTH II in immature salmon were not detectable by RIA whereas plasma GTH I levels were approximately 0.62 0.12 and 0.78 0.13 ng/ml in male and female fish, respectively. Highly purified coho salmon GTH I and GTH II stimulated testicular testosterone production and ovarian estradiol productionin vitro in a similar manner, though GTH II appeared more potent than GTH I. Therefore, it appears that although the salmon pituitary contains predominantly GTH I prior to puberty, the gonad can respond to both GTH I and GTH II.

Abstract: Circulating concentrations of estradiol (E2), vitellogenin (VTG), thyroxine (T4), triiodothyronine (T3) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E2 and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T4 and T3 were relatively constant in winter and spring, and decreased in July. Plasma concentration of T4 increased in November when the fish returned to fresh water. Plasma T3 levels remained low during the autumn. Both T4 and T3 levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.

Abstract: The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

The first piscine follicle-stimulating hormone (FSH) was identified in salmon more than 20 years ago and has since been identified in numerous species of bony and cartilaginous fish. Yet, knowledge of FSH function in fishes is limited because of the lack of highly purified native protein and specific immunoassays for physiological studies in divergent species. The long-term aim of our research is to produce recombinant piscine FSH analogs that can be used to investigate FSH function and enhance reproduction of divergent fish species that are important for aquaculture. Initially we focused on production of recombinant salmon (s)FSH because of the availability of: native sFSH for comparison; cDNAs encoding sFSH, luteinizing hormone (sLH), and thyroid-stimulating hormone (sTSH) subunits and their receptors (R); and sFSH immunoassays and bioassays for characterization of the analog. Our strategy has been to produce recombinant single chain sFSH analogs in either COS-7, CHO, or HEK293T cells and screen them for their ability to induce cAMP production in transiently (HEK293T) or stably (CHO) transfected cells expressing recombinant sFSHR. Analogs that were active in the receptor assays were then tested for steroidogenic activity in vitro using salmon ovarian follicles and immunological cross-reactivity in the sFSH radioimmunoassay (RIA). We found that single-chain analogs in which the C-terminal end of the hCG beta-subunit was used as a spacer to link the sFSH beta-subunit to the alpha-subunit were not expressed well. This result was surprising because this strategy is commonly used to express mammalian FSH and LH; however, it is consistent with earlier studies showing that heterodimers having structures similar to sFSH having altered seatbelt latch sites are assembled differently from mammalian FSH. In contrast, a single-chain construct that encoded FLAG-tagged sFSH beta-subunit-glycine-human alpha-subunit was expressed very well by HEK293T cells. The FLAG tag and the human alpha-subunit were included to facilitate quantification of the material in sandwich assays employing a human alpha-subunit monoclonal antibody for capture and a radioiodinated M1 anti-FLAG antibody for detection. This FSH analog was a very potent stimulator of cAMP production by mammalian cells expressing the sFSHR, and concentrations that gave maximal cAMP induction did not elicit a response from cells expressing the sTSHR. Furthermore, this analog had similar potency to native sFSH in the in vitro bioassay and serial dilutions of the sFSH analog were parallel to the native sFSH in the RIA. In other structure/function studies, we found that FSH analogs in which the human alpha-subunit is replaced by the salmon alpha-subunit II or in which the C-terminal end of the sFSH beta-subunit and N-terminal end of the human alpha-subunit can be truncated without destroying its ability to initiate signal transduction in cells that express sFSHR. We have also prepared and tested several other analogs to characterize the structure and function of sFSH. Our results appear to be consistent with the notion that salmon and human FSH interact with different portions of their respective receptors, a phenomenon predicted on the basis of differences in the folding patterns of salmon and human FSH. Research supported by National Research Initiative Competitive Grant no. 2007-35203-18088 from the USDA CSREES.(poster)

The duality of pituitary gonadotropins in fishes has been established largely through molecular characterization of the genes that encode subunits of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In contrast, little is known about the functional duality of piscine LH and FSH due to the lack of sufficient quantities of highly purified native proteins or well characterized recombinant proteins that maintain activity in vivo for physiological studies. In this study, we examined in vivo biological activity of recombinant coho salmon FSH analog (csFSHa) comprised of human glycoprotein hormone alpha subunit and coho salmon FSH beta subunit expressed as a single chain in stably transfected COS-7 cells. Media containing csFSHa was dialyzed and concentrated by ultrafiltration. The concentrations of immunoreactive and bioactive csFSHa (13.5 mg/L) were estimated by comparison to native csFSH standards in a salmon FSH radioimmunoassay and an in vitro steroidogenesis bioassay. Previous studies from our laboratory have suggested that onset of puberty in male salmon is controlled by FSH because plasma levels of FSH increase and LH is non-detectable in plasma at this stage. Thus, to test whether csFSHa could stimulate spermatogenesis in vivo, the analog (1.35 micrograms/fish) was injected intraperitoneally twice a week for 2 weeks into prepubertal male fish (n=8 per group). For comparison, other groups of fish received either saline (control) or a preparation of partially purified native salmon gonadotropins (ppGTH, mixture of FSH and LH, 1.5 micrograms/fish) due to lack of sufficient amounts of highly purified native proteins for in vivo studies. Fish were sampled for blood and tissues 6-8 h after the last injection. Pieces of testis were frozen for analysis of mRNA levels or fixed for histological staging. The mean gonadosomatic index (GSI; gonad weight/body weight x 100) of analog-injected males was 1.5-fold higher than either controls (p < 0.001) or ppGTH-injected fish (p < 0.01). The expression of putative Sertoli cell transcripts, anti-Mullerian hormone (amh) and gonadal-soma derived growth factor (gsdf), which decline with onset of spermatogenesis in salmon, were suppressed by the csFSHa compared to controls (p < 0.05). In contrast, csFSHa had no effect on plasma levels of 11-ketotestosterone (11-KT) or mRNA levels for steroidogenesis-related genes, such as starand hsd3b. This suggests that the change in testis mass induced by the csFSHa may not be mediated by 11-KT. Interestingly, ppGTH increased 11-KT levels 2-fold, had no effect on GSI, and caused a slight, but not significant elevation in testicular hsd3bmRNA levels. The difference in in vivo steroidogenic activity between ppGTH and the csFSHa may be due to the presence of high amounts of LH in the ppGTH preparation. In conclusion, the present study has shown that a recombinant csFSHa elicits biological activity in vivo that is consistent with the proposed role for FSH in onset of puberty in male salmon. The results also indicate that this strategy for production of piscine FSH analogs will be useful for basic physiological studies as well as practical applications for controlling timing of puberty in commercially important species for aquaculture. Research supported by National Research Initiative Competitive Grant no. 2007-35203-18088 from the USDA CSREES.(poster)

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49.(poster)

It is well established in mammals that follicle-stimulating hormone (FSH) plays a major role in regulating oocyte growth and development by modulating the expression of follicle cell and oocyte factors. In fishes, however, little is known about the function(s) of FSH and regulation of early oocyte growth, a period of development that may affect later egg quality and fecundity. In species that exhibit synchronous gonad development (e.g., salmonids, catfish, striped bass), plasma FSH and/or pituitary gonadotropin subunit expression increases significantly during early secondary oocyte growth and continues to rise during vitellogenesis, suggesting FSH may play a role in regulating follicle development during this period. The goal of this study was to gain a fundamental understanding of how FSH acts at the level of the fish ovary by identifying FSH regulated ovarian genes. To examine time course effects of FSH, coho salmon (Oncorhynchus kisutch) previtellogenic ovarian follicles were cultured with or without 500 ng salmon FSH for 12-72 h. Culture media was collected for estradiol-17beta (E2) measurement, ovarian RNA was isolated, and abundance of candidate oocyte and follicle cell mRNAs was measured by quantitative PCR. Medium E2 levels were significantly elevated in response to FSH by 12 h and plateaued after 48 h. Messenger RNAs encoding steroidogenic enzymes, such as steroid acute regulatory protein (star) and 3beta-hydroxysteroid (hsd3b) were upregulated by 12 h, while P450 aromatase (cyp19a1a) showed no change in response to FSH. Other putative follicle cell transcripts such as FSH receptor (fshr) and lipoprotein lipase (lpl) were depressed initially by FSH treatment and later recovered to control levels. Putative oocyte transcripts such as zona pellucida glycoproteins, alveolin, and a C-type egg lectin showed no change over the 72-h experiment. Additional FSH regulated ovarian genes were subsequently identified through construction of four suppression subtractive hybridization (SSH) libraries with RNA isolated from FSH-treated and untreated whole ovarian follicles, as well as follicle/interstitial cell enriched ovarian tissues. Many of the genes identified by SSH are involved in signal transduction, cell cycle regulation, cell growth and proliferation, apoptosis, cholesterol metabolism and steroidogenesis. Experiments utilizing steroidogenesis inhibitors are underway to distinguish ovarian genes directly regulated by FSH versus those regulated by sex steroids produced in response to FSH. As evident by the rapid and significant increase in E2 release and changes in steroidogenic enzyme gene expression within the ovary, we can conclude that FSH strongly influences steroidogenesis of the salmon ovary during early secondary oocyte growth and may be a critical signal for puberty onset. This is the first comprehensive assessment of the biological actions of FSH in the fish ovary. Discoveries will aid in developing methods to reduce common reproductive dysfunctions in aquaculture, such as delayed puberty and poor egg quality. This project was supported in part by National Research Initiative Competitive Grant no. 2007-35203-18082 from the USDA Cooperative State Research, Education, and Extension Service to JAL.(platform)